1535-9778/08/$08.00⫹0 doi:10.1128/EC.00025-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Identification of Mating Type Genes in the Bipolar Basidiomycetous
Yeast
Rhodosporidium toruloides
: First Insight into the
MAT
Locus
Structure of the
Sporidiobolales
䌤
†
Marco A. Coelho, Andre´ Rosa, Na´dia Rodrigues, A
´ lvaro Fonseca,* and Paula Gonc¸alves
Centro de Recursos Microbiolo´gicos, Departamento de Cieˆncias da Vida, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
Received 18 January 2008/Accepted 1 April 2008
Rhodosporidium toruloidesis a heterothallic, bipolar, red yeast that belongs to theSporidiobolales, an order within a major lineage of basidiomycetes, thePucciniomycotina. In contrast to other basidiomycetes, consid-erably less is known about the nature of the mating type (MAT) loci that control sexual reproduction in this lineage. Three genes (RHA1,RHA2, andRHA3) encoding precursors of theMAT A1pheromone (rhodotorucine
A) were previously identified and formed the basis for a genome walking approach that led to the identification of additionalMATgenes in complementary mating strains of R. toruloides. Two mating type-specific alleles encoding a p21-activated kinase (PAK; Ste20 homolog) were found between theRHA2andRHA3genes, and identification inMAT A2strains of a gene encoding a presumptive pheromone precursor enabled prediction of the structure of rhodotorucine a. In addition, a putative pheromone receptor gene (STE3 homolog) was identified upstream ofRHA1. Analyses of genomic data from two closely related species,Sporobolomyces roseus
andSporidiobolus salmonicolor, identified syntenic regions that contain homologs of all the above-mentioned genes. Notably, six novel pheromone precursor genes were uncovered, which encoded, similarly to theRHA
genes, multiple tandem copies of the peptide moiety. This suggests that this structure, which is unique among fungal lipopeptide pheromones, seems to be prevalent in red yeasts. Species comparisons provided evidence for a large, multigenicMATlocus structure in the Sporidiobolales, but no putative homeodomain transcription factor genes (which are present in all basidiomycetousMATloci characterized thus far) could be found in any of the three species in the vicinity of theMATgenes identified.
Fungal mating type (MAT) loci are specialized genomic re-gions that determine cell type identity and coordinate the sex-ual cycle. Their genetic structure has been relatively sparsely sampled in broad phylogenetic terms, but results of published studies have revealed both conserved features and remarkable diversity in ascomycetes and basidiomycetes (8, 13). A com-mon trait of fungalMATloci is the presence of genes encoding homeodomain or other classes of transcription factors that control the expression of genes that establish cell type identity and activate sexual development. In basidiomycetes, two major types ofMATloci have so far been recognized (9). The tet-rapolar mating system of the corn smutUstilago maydisand the mushroomsCoprinopsis cinereaandSchizophyllum communeis governed by two small (⬍10-kb) unlinked loci: one encodes homeodomain transcription factors (HD1andHD2homologs), and the other encodes lipopeptide pheromones and phero-mone receptors (STE3homologs), which mediate intercellular signaling prior to cell fusion and/or sexual development after mating (9). Each locus may be multiallelic, which can result in up to thousands of compatible mating types in certain tetrapo-lar species. On the other hand, in the bipotetrapo-lar system of the human pathogen Cryptococcus neoformansand of the barley
smutUstilago hordeithe twoMATloci described above for the tetrapolar species are physically linked, forming a single, large (⬎100-kb) multigene locus, which contains additional genes that may or may not be related to mating (5, 25). In this case there are only two possible combinations ofMATgene alleles, and hence only two compatible mating types exist. A unique feature ofC. neoformansis that genes of three components of the pheromone-activated signaling cascade (viz., STE11,
STE12, andSTE20) are part of theMATlocus and, therefore, mating type specific (25).
Three major lineages are currently recognized in the Basid-iomycota(18). One of them, the subphylumPucciniomycotina, comprises the rust fungi (e.g., the wheat rustPuccinia graminis, whose genome annotation was recently released [http://www .broad.mit.edu/annotation/genome/puccinia_graminis]) and other plant parasites, such as the anther smuts (Microbotryum
spp.), as well as many saprobic yeast taxa. The latter include the so-called “red yeasts” in the generaRhodosporidiumand
Sporidiobolus, which are classified in the orderSporidiobolales
(30). Fungi in this lineage have remained virtually unexplored as to the content and organization of theMATloci. However, preliminary work with the bipolar anther smutMicrobotryum violaceumhas shown that chromosomes carrying theMATloci are sexually size dimorphic and rich in repetitive sequences (20). A recent study based on expressed sequence tag data (39) provided the first clues on their gene content, but the respective genetic organization is yet to be elucidated. The remaining two basidiomycetous subphyla contain the few taxa whose MAT loci have been investigated in detail (9): the
* Corresponding author. Mailing address: Centro de Recursos Mi-crobiolo´gicos, Departamento de Cieˆncias da Vida, Faculdade de Cieˆn-cias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Phone and fax: 351212948530. E-mail: amrf@fct.unl.pt.
† Supplemental material for this article may be found at http://ec .asm.org/.
䌤Published ahead of print on 11 April 2008.
evolution ofMATloci in basidiomycetes.
As experimental models, saprobic basidiomycetous yeasts have a major advantage over their strictly filamentous or par-asitic counterparts because they complete their life cycle on culture medium (11). The vast majority of sexual yeast taxa, such asRhodosporidium, have dimorphic life cycles: upon fu-sion of compatible cells, elicited by the mating pheromones, the haploid yeast phase switches to a dikaryotic filamentous phase, during which basidia are eventually produced. In
Rhodosporidium basidia arise from teliospores, which are thick-walled, resting cells that form on the dikaryotic hyphae and where karyogamy takes place (11). Germination of basidio-spores restores the yeast phase.
All knownRhodosporidiumspecies were isolated as haploid yeasts and have a bipolar mating behavior, i.e., their strains belong to either one of two complementary mating types, des-ignatedA1and A2orA anda(1, 10). Based on our current understanding of basidiomyceteMATloci, it is hypothesized that the genes encoding the pheromones, the pheromone re-ceptors, and the homeodomain transcription factors are part of a single MAT locus in Rhodosporidium species. Pioneering work by Japanese researchers led by S. Fukui in the late 1980s identified three genes encoding the pheromone precursors in a mating typeA1(orA) strain ofR. toruloides(2). These genes were not present in a complementary mating typeA2 (ora) strain (3) and have been suggested to be part of theMATlocus (6). Fukui and coworkers (1) had previously characterized the early stages of the mating process inR. toruloides and sug-gested thatA1cells constitutively produced a pheromone (A
factor, later named rhodotorucineA), which induced the pro-duction ofafactor byA2cells. The studies of Akada et al. (2, 3) were not continued, and no additional information on the
MATloci ofRhodosporidiumspecies was published since then. Here we present evidence that the gene encoding a p21-activated kinase (PAK; Ste20-like) possibly involved in phero-mone signaling is mating type specific and that, along with the multiple pheromone precursor genes, it belongs to theR. toru-loides MAT locus. We also found that this genomic region displays a high level of synteny with homologous genomic re-gions from the closely related yeast Sporobolomyces roseus
(11), whose genome sequence has been recently released (Joint Genome Institute [JGI]; http://genome.jgi-psf.org /Sporo1/Sporo1.home.html). Genomic fragments of a close rel-ative of the latter species,Sporidiobolus salmonicolor(11), are also available and provided an additional source of sequence data for comparison. Analysis of our data on R. toruloides
together with that of theS. roseus genome and of theS. sal-monicolor Trace Archive provided evidence as to the MAT
locus structure of members of theSporidiobolalesand consti-tutes the second instance of the presence of a PAK kinase gene within a basidiomyceteMATlocus.
microscopically after 1 week for the production of mycelia with clamp connec-tions and teliospores, using phase-contrast optics.
An enlarged set of strains was used to check mating type specificity of pher-omone genes by PCR, in addition to the four strains mentioned above, namely, CBS 350, CBS 315, MSD 231, A421, and A415 (MAT A1); CBS 349, PYCC 4786, PYCC 4943, PYCC 5109, PYCC 5081, A412, A401, A413, and A456 (MAT A2). MSD and A strains are environmental isolates obtained by Gadanho et al. (16). Miscellaneous.Isolation of genomic DNA was performed essentially as de-scribed by Sampaio et al. (29), with a few modifications. After centrifugation, the cell extracts were submitted to digestion with proteinase K (1 mg/ml; 1 h, 37°C) and, subsequently, to phenol (pH 8.0) and chloroform-isoamyl alcohol (24:1) extractions. Nucleic acids were dissolved in 100l of distilled water after pre-cipitation.
PCRs were performed in a final volume of 25l and contained the following components (unless stated otherwise): 2 mM of MgCl2, 0.25 mM of each of the
four deoxynucleoside triphosphates (GE Healthcare), 0.8M of primer, 5l of template (genomic DNA was diluted 1:750), and 1 UTaqDNA polymerase (Fermentas, Canada). Thermal cycling consisted of a 5-minute denaturation step at 95°C, followed by 35 cycles of denaturation at 94°C for 30 s, 30 s at the annealing temperature (variable), and extension at 72°C (variable time); the annealing temperatures and extension times used in each reaction, as well as the sequences of all the primers, are listed in Table S1 of the supplemental material. A final extension of 7 min at 72°C was subsequently performed.
Amplification products were purified using the GFX PCR DNA and gel purification kit (GE Healthcare) and either cloned into the pMOSBluevector with the pMOSBlueblunt-ended cloning kit (GE Healthcare) and sequenced or sequenced directly (sequencing was performed by STABVida, Portugal).
Mating type specificity ofRHAgenes.TheMAT A1specificities of theRHA1,
RHA2, andRHA3pheromone precursor genes were checked by PCR amplifica-tion with gene-specific primers based on the sequences determined by Akada et al. (2) and using genomic DNA from bothMAT A1andMAT A2strains as template. One forward primer common to the three genes (RHAFw) was used together with one reverse primer specific for each gene (RHA1Rev, RHA2Rev, and RHA3Rev) (see Table S1 in the supplemental material).
Following the identification of theRHA2.A2gene, mating type specificity was checked in an enlarged set of strains using reverse primers specific for either the
RHA2or theRHA2.A2coding regions (MC075 and MC071, respectively) (see Table S1 in the supplemental material) and a common forward primer based on a conservedSTE20region in the two mating types (Ste20Dw2A2) (see Table S1 in the supplemental material).
Genome walking.A genome walking approach using the Universal Genome Walker kit (BD Biosciences Clontech) was employed to characterize theMAT
loci ofR. toruloides.This method consists of preparing a set of genomic DNA libraries each requiring the ligation of adapters to the ends of restriction frag-ments obtained by complete digestion with a particular enzyme (blunt cutters). These libraries are subsequently screened using combinations of gene-specific and adapter primers (AP1 and AP2). For bothMAT A1strain PYCC 4416 and forMAT A2strain PYCC 4661, the genomic libraries were prepared using EcoRV, PvuII, and StuI. Each Genome Walker library was used as a template in independent amplification reactions. Primer design and PCR conditions fol-lowed the manufacturer’s instructions. The fragment upstream of theRHA2gene (fragment 1; Fig. 1) was obtained from the EcoRV library, whereas the two fragments encompassing part of theMAT A2 STE20gene (fragments 5 and 6; Fig. 1) were amplified from the StuIMAT A2library.
the remaining strains under study (MAT A1andMAT A2). The PCR products were purified and directly sequenced by primer walking.
PCR amplification of a putativeMAT A1 pheromone receptor (STE3 ho-molog).For the identification of pheromone receptor genes inRhodosporidium toruloides, thercb1gene fromCoprinopsis cinereus(accession number Y11082) was used to perform a Tblastx search (4) in theS. roseusgenome sequencing project database (JGI; http://genome.jgi-psf.org/Sporo1/Sporo1.home.html). Positive hits were retrieved and used to search the NCBI Trace Archive se-quences ofS. salmonicolor, yielding an assembled sequence for the SsSTE3gene. The SsSTE3and SrSTE3genes were in turn aligned with other basidiomycete pheromone receptor genes (PDSTE3.3gene fromPleurotus djamor[AY462110],
bbr1fromSchizophyllum commune[U74495], and STE3afromCryptococcus neoformansvar.neoformans[XM_570116]), and the conserved regions were used to design degenerate primers (NCAR3Fw and NCAR4Rev) (see Table S1 in the supplemental material). These degenerate primers were used in PCRs with genomic DNA fromR. toruloidesbut failed to yield an amplification product. A similar reaction using as template genomic DNA from a closely related species (Rhodosporidium kratochvilovaePYCC 4818;MAT A1) (29) produced an ampli-con which was directly sequenced and found to encode a partial open reading frame (ORF) of aSTE3-like pheromone receptor. The new sequence was used to refine the design of a second set of primers forR. toruloides. These primers (NCAR5STE3Fw and NCAR6STE3Rev) (see Table S1 in the supplemental material) permitted the amplification of part of theSTE3homolog inR. toru-loides. The position and orientation of theSTE3homolog relative to theRHA1
gene in R. toruloides were checked by PCR with gene-specific primers (NCAR5STE3Fw and RHA1Rev) (see Table S1 in the supplemental material). PCRs were performed in a final volume of 50l and contained the following components: 1 Long PCR buffer, 2 mM of MgCl2, 0.2 mM of each of the four
deoxynucleoside triphosphates (GE Healthcare), 1.0% dimethyl sulfoxide, 1.0
M of primer, 5l of template (genomic DNA diluted 100-fold), and 2.5 U Long PCR enzyme mix (Fermentas, Canada). Thermal cycling consisted of a 3-minute denaturation step at 94°C, followed by an initial round of 10 cycles of denatur-ation at 95°C for 20 s, annealing at 60°C for 30 s, and extension at 68°C for 6 min and a second round of 25 cycles, increasing the extension time 2 s in each cycle. A final extension of 10 min at 68°C was performed. The nucleotide sequences of the ends of the amplified fragment were obtained by direct sequencing using the same primers.
In silico analyses.Primers were designed using Primer Premier 5.0 software (Premier Biosoft International). The assembly of DNA sequences was per-formed using SeqMan II software (v.6.1) from DNASTAR. Gene models were constructed by combining nucleotide homology searches in the NCBI database, using Blastn and tBlastx (4) and in silico predictions of putative introns and coding sequences using AUGUSTUS software (http://augustus.gobics.de /submission) (34). Blastp searches in NCBI were used to check for conserved
domains within the deduced amino acid sequences and to assign putative gene functions.
The genomic DNA sequences ofS. roseus(IAM 13481) showing a high degree of identity to theSTE20homolog ofR. toruloidesPYCC 4416 (MAT A1) were retrieved from the first release of theS. roseusgenome sequencing project by JGI (http://genome.jgi-psf.org/Sporo1/Sporo1.home.html; scaffold 9, contig 11). An-notation of the 40-kb region encompassing theSTE20gene was completed using Tblastx. The NCBI Trace Archive sequences ofS. salmonicolor(IAM 12258) contained sequences with a high percent sequence identity to that of theSTE20
gene ofS. roseus. The relevantS. salmonicolorfragments were assembled step-wise from theSTE20region. Following homology searches using the SsRHA2
gene, the SsRHA1and SsRHA3genes ofS. salmonicolorwere also retrieved and assembled (the Trace Archive sequences used are listed in Table S2 of the supplemental material). Nucleotide and amino acid sequences were aligned using ClustalW (37) and T-Coffee (28), respectively.
Tblastx was used to search the entire genome ofS. roseusfor homologs of the homeodomain transcription factors (HD1 and HD2).
Nucleotide sequence accession numbers.The accession numbers for the novelR. toruloidesDNA sequences are as follows: EU386160 (RtSTE20.A1, PYCC 4416, fragments 1 and 2) (Fig. 1); EU386161 (RtSTE20.A2 and
RHA2.A2, PYCC 4661, fragments 3 to 6) (Fig. 1); EU401861 and EU401862 (RtSTE20.A1, PYCC 5082, and RtSTE20.A2, PYCC 4417, respectively; region corresponding to the fragments 3 and 4) (Fig. 1); EU401863 (RtSTE3.A1, PYCC 4416, partial sequence).
RESULTS
MATstatus ofR. toruloidesstrains.To characterize genomic regions involved in mating inR. toruloides, the four strains to be studied (two of each mating type) were tested for their ability to mate and form hyphae with clamp connections and teliospores, characteristic of the sexual cycle in this yeast (10). All four strains were fertile,MAT A1isolates mated only with
MAT A2isolates, and none of the strains were self-fertile. The mating results were in accord with the presence of the phero-mone precursor genes previously described by Fukui and co-workers (2) (RHA1, RHA2, andRHA3), since they were de-tected by PCR with gene-specific primers only in strains that mated asMAT A1in crossing experiments.
Genomic regions in the vicinity of the pheromone precursor genes inR. toruloides MAT A1.Two of the pheromone precur-sor genes previously identified (RHA2andRHA3) were found to be located close to each other on opposite DNA strands (2) (Fig. 1), while the location of the third gene (RHA1) in relation to the former remained to be established. Since in basidiomy-cetes the genes encoding pheromone precursors have so far been found almost invariably inside theMATloci, we used the sequences of the threeRHAgenes as anchors for a genome walking approach to explore the adjacent regions, presumed to be part of theMAT A1locus in this yeast.
To this end, a genome walking approach was designed that resulted in the amplification of a 0.7-kb genomic DNA frag-ment upstream of theRHA2 gene (Fig. 1, fragment 1). Se-quencing of this fragment revealed two notable features: first, a region of 204 bp located immediately upstream of theRHA2
gene was found to be identical to the corresponding region upstream of the RHA3gene sequenced by Akada et al. (2); second, a region of approximately 200 bp at the 5⬘end of the fragment turned out to encode a peptide with strong similarity to the catalytic domain of PAK kinases (19). To investigate whether this was part of a PAK kinase gene, the entire inter-vening genomic region between theRHA2and RHA3genes was amplified and sequenced by primer walking (Fig. 1, frag-ments 1 and 2). This region was indeed found to contain a putative PAK kinase gene (named RtSTE20), homologous to genes found in, for example, Ustilago maydis, Cryptococcus neoformans, and Pneumocystis carinii (32, 33, 38). In silico analysis predicted the presence of at least four introns in this gene.
The RtSTE20 gene is mating type specific in R. toruloides MAT A1. Since the RtSTE20 gene is located between two mating type-specific genes, we anticipated that it would be part of the MAT A1 locus. In this case, its sequence would be expected to exhibit mating type-specific polymorphisms. To investigate this, the RtSTE20 gene was amplified and se-quenced almost completely in the four strains under study (the regions sequenced, approximately 1.7 kb, correspond to frag-ments 3 and 4 depicted in Fig. 1). The sequences were indeed found to be almost identical between alleles obtained from strains of the same mating type (eight differences between the two MAT A1strains and three differences between the two
MAT A2strains). However, sequences obtained for the oppo-site mating types had approximately 80 single nucleotide dif-ferences, which would lead to nine amino acid changes in the conserved domains of the RtSte20 protein (see Fig. S1 in the supplemental material). The large majority of the nucleotide differences between the two alleles were, however, located in less-conserved regions of the RtSTE20gene. This observation supports the conclusion that RtSTE20is a mating type-specific gene inR. toruloides.
A putative pheromone precursor gene inR. toruloides MAT A2.
To explore the organization of theMAT A2locus ofR. toru-loides, the region around the RtSTE20gene inMAT A2strain PYCC 4661 was analyzed by genome walking. A fragment of 2 kb downstream of the STE20 gene was amplified and se-quenced (Fig. 1, fragment 5). A completely dissimilar sequence was found downstream of the RtSTE20 gene in theMAT A2
strain when compared to the same region in aMAT A1strain (encoding the RHA2 gene). To ascertain if this region also encoded a pheromone precursor, the sequence was examined for the presence of an open reading frame exhibiting charac-teristics in keeping with the general features of previously characterized pheromone precursor genes (6, 7). Indeed, a region was found potentially encoding two identical copies of a 13-amino-acid peptide and exhibiting at the C terminus a se-quence of four amino acids (CAAX motif) very similar to that of theMAT A1pheromone precursors (Fig. 2 and 3). The most likely initiation codon is preceded by two CT-rich regions (Fig. 2), one of which is identical to that found upstream of the transcription initiation site of theRHA(MAT A1) genes (2). Therefore, we postulate that this novel gene, namedRHA2.A2, encodes a precursor of the peptide moiety of theR. toruloides MAT A2pheromone (rhodotorucinea).
Mating type specificity of RtSTE20/RHAregions.In order to check linkage between mating behavior and the presence of the newly identifiedRHA2.A2/STE20region, a specific primer based on theRHA2.A2coding region was used in aMAT A2
diagnostic PCR in combination with a RtSTE20 forward primer. A total of 14 additional strains (fiveMAT A1and nine
MAT A2) whose mating behavior was previously checked were tested for the presence of the putative MAT A2 locus se-quences. As expected, an amplicon was obtained only forMAT A2 strains (results not shown). Conversely, when the same
RtSTE20primer was used in combination with a primer based on theRHA2(MAT A1) coding sequence in a PCR assay with the same strain set, onlyMAT A1strains yielded an amplifica-tion product (results not shown). These results strongly suggest that this region is indeed part of theMATlocus ofR. toruloides.
Notably, oneR. toruloidesisolate (A399) (16), which was not included in the previous strain set because it exhibited ambig-uous mating behavior (apparent sexual compatibility with sev-eralMAT A1andMAT A2strains), tested positive in bothMAT A1andMAT A2diagnostic PCR assays, although amplification ofMAT A1sequences was weaker than observed forMAT A1
strains.
Direct evidence of linkage of the characterized genomic region inR. toruloidestoMATby genetic analysis of F1progeny
was not possible since basidiospores form only upon germina-tion of teliospores embedded in the agar medium and are thus not easily subjected to micromanipulation.
Comparison with syntenic genomic regions from related red yeasts. A search in the recently released genome data of
Sporobolomyces roseusfor genes with a high percent sequence similarity with the catalytic domain of RtSTE20 identified a gene likely to represent a RtSTE20 homolog. Notably, this gene (named henceforth SrSTE20) is also flanked by two di-vergently transcribed putative pheromone precursor genes, which were named SrRHA2and SrRHA3, based on synteny with theRHA2andRHA3genes fromR. toruloides.Unlike the latter, the two genes fromS. roseusare completely identical not only in the coding sequence but also in the promoter and terminator regions. Furthermore, a third identical gene (named SrRHA1) was found downstream of a gene probably encoding a 30- to 40-kDa subunit of RNA polymerase III (Pol III). Notably, the 3⬘ end of the coding sequence of the R. toruloideshomolog of this Pol III subunit was found upstream of theRHA1gene in the genomic DNA fragment sequenced by Akada et al. (2).
For Sporidiobolus salmonicolor, genomic sequences are available as Trace Archives (short unassembled genomic DNA fragments). A search of this database revealed the presence of a gene very similar to theSTE20 genes of S. roseus and R. toruloides. This gene (SsSTE20) was used as an anchor to assemble a contiguous fragment that extended to approxi-mately 2 kb downstream of its 3⬘end, where a putative pher-omone precursor gene (named SsRHA2) was similarly found (Fig. 4). The sequence of the SsRHA2gene was used in new searches in the Trace Archives and led to the identification of two additional genes potentially encoding pheromone
precur-sors. While for one of the genes (SsRHA3) the longest possible contiguous fragment did not provide any clue as to its location, the second gene (SsRHA1) was found downstream of a partial ORF encoding the 30- to 40-kDa subunit of RNA polymerase III (Fig. 4). The SsRHA1and SsRHA3coding sequences are completely identical.
InS. roseusa long contiguous sequence around the SrSTE20
gene is available. Therefore, in order to find out whether more
MAT-related genes could be found in the vicinity of this SrSTE20 core, a genomic region of approximately 40 kb was scrutinized for the presence of ORFs. Notably, a STE3-like gene (17), potentially encoding a pheromone receptor, was found close to the SrRHA1gene (Fig. 4, upper panel). Many other genes, apparently unrelated to mating but exhibiting a high percent sequence similarity with genes identified in other organisms, were also found in this region and are indicated in Fig. 4 and listed in Table S3 of the supplemental material. These results suggest that the entire region between the pher-omone receptor core and the SrSTE20core may be part of a large MAT locus in this yeast. A STE3-like gene was also identified in S. salmonicolor, but the incompleteness of the genome sequence data did not allow any inference about its location with respect to the otherS. salmonicolorgenes iden-tified.
A pheromone receptor gene in Rhodosporidium. The two
STE3-like genes identified in S. roseus and S. salmonicolor
share regions of considerable sequence identity. Using a first pair of degenerate primers based on these regions, we were unable to amplify aSTE3fragment fromR. toruloides. How-ever, it was possible to obtain an amplicon when genomic DNA fromRhodosporidium kratochvilovae, a species closely related to R. toruloides (29), was used as a template in the PCR. Sequencing of this fragment and its alignment with the above-mentioned STE3-like genes (results not shown) allowed the design of another set of primers that led to the amplification of the correspondingSTE3fragment fromR. toruloides MAT A1
strain PYCC 4416, as confirmed by direct sequencing. Ampli-fication of the latter fragment was possible only in MAT A1
strains, as expected for a mating type-specific pheromone re-ceptor gene.
PCR with RtSTE3 and RHA1 gene-specific primers (see Table S1 of the supplemental material) yielded an amplifica-tion product of approximately 5 kb, consistent with a gene organization identical to that found inS. roseus.Sequencing of the ends of the amplified fragment confirmed the suspected
synteny, including the presence of the gene encoding the RNA Pol III subunit between theSTE3andRHA1genes (Fig. 4B).
Search for putative HD homologs inS. roseus.We found a gene encoding a putative HD1 homolog in theS. roseus ge-nome (scaffold 7, contig 11). Blastp searches in fungal protein databases using part of the sequence of the encoded protein retrieve most of the basidiomycetous HD1 proteins previously characterized. No HD2 candidates with such characteristics were found, although additional genes potentially encoding homeodomain proteins were identified in the S. roseus ge-nome.
The gene encoding the putative HD1 homolog was recently annotated in the S. roseus genome, but at this stage of the assembly of the sequence data the distance to theSTE20/RHA
region cannot be precisely determined. However, even if they lie on the same chromosome, the distance of the SrSTE20core to thisHD1candidate would be necessarily in excess of 600 kb, as judged from the positions of the genes within the scaffolds; the shortest distance to one of the ends of the respective scaffolds is 213 kb (STE20scaffold) and 410 kb (HD1scaffold), respectively. The region around this putative HD1-like gene was carefully screened for the presence of aHD2 homolog, since in most basidiomycetous MATloci the HD1 and HD2
genes are closely linked and divergently transcribed. However, noHD2 candidate gene was found, even when the homology searches using tBlastx were extended to the entire genome. Nevertheless, it should be noted that the presence of introns
may considerably complicate this kind of search based on a short conserved region, as for example in the case of theSxi2a
gene (HD2homolog) ofCryptococcus neoformans(21).
DISCUSSION
In this paper we present the first insight into the structure of the MATlocus of a group of fungi that has remained unex-plored in this respect. While the basis of the work consisted of the identification of novel genomic regions inRhodosporidium toruloides, we have also analyzed syntenic genomic regions in two additional species belonging to the Sporidiobolales, namely,Sporobolomyces roseusandSporidiobolus salmonicolor. We found that two of theR. toruloidespheromone precursor genes previously characterized by Akada et al. (2) flanked a gene potentially encoding a kinase of the PAK family, whose homologs were also present in theS. roseusandS. salmonicolor
genomes. These three PAK kinase orthologs encode proteins that possess the two functional domains common to the entire PAK kinase family, namely, a highly conserved C-terminal catalytic domain and a CRIB domain (cdc42/ras interaction binding domain) (see Fig. S1 in the supplemental material). In addition, they have a third domain (PH-pleckstrin homology) (see Fig. S1 in the supplemental material) that is present in a subset of the PAK family members, such as the Ste20 kinases of Cryptococcus neoformans and C. gattii (38) and the Cla4 kinase of Ustilago maydis (26). The former proteins were
shown to be involved in mating or required for the onset of the filamentous phase and are present in theMATloci of the two
Cryptococcussibling species. However, theMAT-specific alleles ofSTE20have diverged considerably more inC. neoformans
than inR. toruloides(approximately 70% versus 90% sequence identity) (Fig. 5). It is therefore unlikely that the twoR. toru-loidesproteins exhibit functional dissimilarities comparable to those found for theC. neoformans MAT-specific kinases (38). The rhodotorucineA(RHA) genes were the first pheromone precursor genes whose structure was determined in basidiomy-cetes (2). The same authors (23, 24) had previously determined the chemical structure of the mature pheromone as a lipopep-tide (farnesyl-undecapelipopep-tide), in which the C-terminal cysteine of the peptide moiety was shown to be covalently linked to a farnesyl group through a thioether bond. That was the first demonstration of this type of posttranslational modification, which was subsequently found in many other fungal phero-mones, such as theafactor ofSaccharomyces cerevisiae, as well as in other eukaryotic proteins (6, 7).
Like other prenylated proteins, the predicted rhodotorucine
Aprecursors contain a C-terminal CAAX motif (2), where A stands for an aliphatic amino acid and X for any amino acid (often an alanine in fungal pheromones) (7). The C-terminal motif of the rhodotorucine A precursor is actually CTVA, where threonine does not conform to the general pattern of fungal lipopeptide pheromones, which have either valine, iso-leucine, or leucine as the middle amino acids (7, 27). However, recently Schirawski et al. (31) predicted six pheromone pre-cursors in the smutSporisorium reilianum, four of which have a terminal CTIA motif. The rhodotorucineA precursors are, nevertheless, unique among fungal lipopeptide pheromones, since they contain multiple tandem copies of the peptide moi-ety (2, 7). Akada et al. (2) suggested that those multiple re-peats could have originated by an unequal crossing-over mech-anism. The same authors (2) also affirmed that the production of multiple peptides per mole of precursor (as well as the presence of multipleRHAgenes) may increase the production of the mating pheromone per cell and that this may be a prerequisite for the poorly diffusible small lipophilic peptides to function effectively. In fact, the mating reaction inR. toru-loidesappears to be triggered by the production of rhodotoru-cineAbyMAT A1(orA) cells, according to the chain of events suggested by Abe et al. (1). The other unusual trait of the rhodotorucineAprecursors is that CAAX motifs are present between the repeats, where they are followed by a lysine res-idue, which has been proposed as a signal for proteolytic pro-cessing (2, 6).
Our findings provide interesting new insights into this topic. On one hand the putative rhodotorucineaprecursor predicted from the RHA2.A2 gene sequence appears to have also a CAAX C-terminal motif (CTIA instead of CTVA) but only two repeats of the peptide moiety, which are apparently pre-ceded by a much longer N terminus tail and separated by a longer peptide spacer (Fig. 3). These differences suggest that the pheromone precursor genes of the two opposite mating types ofR. toruloidescould be processed by different mecha-nisms. On the other hand our analyses of theS. roseusandS. salmonicolorgenome data revealed the presence of three pu-tative pheromone precursor genes which resemble those ofR. toruloides MAT A1(Fig. 3). The presence of three pheromone precursor genes in the three species examined, each encoding three to six copies of the mature peptide moiety, suggests that they have in common the production of abundant pheromone as an important feature of the mating process. This could mean that compatible partner recognition occurs mainly or exclu-sively through pheromone-receptor interactions. This is in con-trast with tetrapolar species, likeU. maydis, where mate rec-ognition requires a second level of compatibility determined by the multiallelic mating typeblocus, which encodes the bE and bW homeodomain transcription factors.
One striking difference between theRHAgenes ofR. toru-loides and those found in the other two species is that the C-terminal motifs of the latter appear not to correspond to CAAX boxes. However, a lysine residue is present in the C terminus of theS. roseus and S. salmonicolorprecursors im-mediately after a putative CAAX box and may signal a pro-teolytic processing site that could produce a CAAX-bearing intermediate. In all three sporidiobolaceous species, the inter-nal CAAX boxes (as well as the putative termiinter-nal CAAX
receptor gene region and a region encoding homeodomain transcription factors (5, 25). We did not come across candidate homeodomain transcription factor genes upon inspection of a 40-kb genomic region surrounding the pheromone precursor and pheromone receptor genes inS. roseus, but a very likely
HD1candidate was found at a distant location in the genome. This could mean that theMATlocus extends beyond the region inspected inS. roseusor that the homeodomain transcription factors are not linked to the MAT locus in this species, a situation corresponding to the Aon MATstatus proposed by Fraser et al. (15) as one of the possible modes of transition from tetrapolar to bipolar mating systems. The other instance of a bipolar basidiomycete whose MAT locus structure has been elucidated, the mushroomCoprinellus disseminatus, is an example of the reverse situation with respect to MATlocus content, since in this case mating type identity is conferred by a short locus containing one or two pairs of closely linked homeodomain transcription factor genes but the pheromone and pheromone receptor genes are not mating type specific (9, 22). However, it should be kept in mind that althoughS. roseus
is the only species in theSporidiobolalesfor which the entire genome is available for scrutiny, it has been deemed an asexual species and therefore itsMAT gene organization cannot be unequivocally linked to a functional sexual cycle. On the other hand, the sequencedS. roseusstrain may well correspond to a haploid mating type of a yet-unrecognized sexual species, a situation frequently encountered among basidiomycetous yeasts (11). In favor of the latter hypothesis is the fact that none of the putativeS. roseus MATgenes examined showed any sign of being degenerated or nonfunctional. On the other hand, S. salmonicolor is a heterothallic species, and the se-quenced strain (IAM 12258, CBS 483) has a known mating status (MAT A2) (35).
Putative pheromone receptor genes (STE3homologs) with a high degree of sequence identity were found inR. toruloides MAT A1strains and in the other two sporidiobolaceous spe-cies. In R. toruloides and S. roseus the genes appear to be located upstream of theRHA1gene, with the same gene un-related to mating (a subunit of RNA Pol III) in between. Therefore, also in this region, located inS. roseusat a distance of approximately 10 kb from the SrSTE20core, a strict synteny occurs between the three species (Fig. 4). Since the pheromone receptor gene remains to be identified inMAT A2strains ofR. toruloides, it is so far unknown whether theMAT A2locus is also syntenic in this region. The strong sequence similarity observed between the Ste3 proteins and even between the putative mature pheromone regions in the three species sug-gests that the threeMATloci depicted in Fig. 3 represent the “same mating type,” analogous toMAT A1inR. toruloides. The limited amount of information available for the opposite mat-ing type (restricted to the RtSTE20.A2andRHA2.A2 genes in
R. toruloides) precludes any conclusion with respect to
ex-sporidiobolaceous species is in sharp contrast with the highly variableMATgene order and orientation observed in the Cryp-tococcusspecies. Future studies will be aimed at a more com-plete definition of theSporidiobolales MATloci, with special emphasis on the identification of additionalMATgene candi-dates, determination of the full extension of theMATloci, and investigation of the role of homeodomain transcription factors in sexual reproduction. The recent release of genomic data from two other fungi in thePucciniomycotina(Puccinia graminis
andMicrobotryum violaceum) (39) and the future completion of theRhodosporidium babjevaegenome (an ongoing sequenc-ing project at the JGI) will also contribute to placsequenc-ing our findings in a broader context and help to retrace the evolution of theMATloci in this basidiomycete subphylum.
ACKNOWLEDGMENTS
The work was partially supported by project POCTI/BME/44322/ 2002 and Ph.D. grant SFRH/BD/29580/2006 (M.C.) from the Portu-guese Foundation for Science and Technology.
We thank Kenneth Wolfe (Smurfit Institute of Genetics, University of Dublin) for providing access to theSporobolomyces roseusgenome data before it was publicly available and Jose´ Paulo Sampaio (CREM, Universidade Nova de Lisboa) and coworkers for the R. toruloides strains.
ADDENDUM IN PROOF
The strain sequenced by the JGI (IAM 13481), labeled as Sporobo-lomyces roseus, does not actually belong to that species, as was recently shown by Vale´rio et al. (Int. J. Syst. Evol. Microbiol. 58:736–741, 2008). It corresponds to an unnamed taxon with an unknown sexual status (see Fig. 2 of the above-mentioned paper).
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